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Aim of this study is to define the groundwater flow of thermal area of Saturnia, through both chemical and isotope analyses. After a first detailed geological-geophysical survey and a quantification of the available groundwater resources, we analyzed several chemical compounds (Na, Ca, Mg, K, Chlorides, Sulfates, Nitrates, total alkalinity, Li, Sr, B, Fluorides, soluble Silica, free Carbon Dioxide, Sb, As, Se, H2S, Fe, Mn, Hg e Pb) and isotopes (2H; 3H, 3He, 4He, 13C, 18O, 86Sr, 87Sr). Excluding the uncertainties mainly related to a large repetition of measures over time, we concluded the following conclusions. Both Lithium and Boron high values and 87Sr/86Sr values highlighted that “Calcare Cavernoso” limestone geological formation constitutes the Saturnia thermal aquifer. Such samples showed both the same chemical characteristics and source area. Boron, Lithium, δ2H, δ18O and 87Sr/86Sr show the Saturnia thermal aquifer is separated by those of Pitigliano (Latera- Bolsena thermal circuit) and Bagni San Filippo (Mt. Amiata thermal circuit). 18O, 3H e δ13C measured in samples coming from Saturnia thermal spring respectively indicate that water feeding the thermal aquifer infiltrate at 350-440 m a.s.l., that they are almost 30 years old, and finally they that are isolated regarding to external infiltration. Low value of R/Ra tends to exclude the influence of Earth mantle to the geothermal anomaly of Saturnia area. The high content in Selenium indicates a possible interaction between Saturnia spring water and those coming from the Saturnia well, with volcanic fluids. However, considering also the evidence of the separation between the Saturnia groundwater flow and those of neighboring thermal fields (Bolsena and Mt. Amiata), we hypothesized that only a exchange among these volcanic complexes and the source area of thermal water points exists, but not among these complex and Saturnia aquifer.